Functional and inflammatory disorders of the gut are characterized by allodynia (the perception of a normally non-painful stimulus as painful) and hyperalgesia (the enhanced perception of pain). The development of visceral hyperalgesia and its underlying central sensitization relies crucially on the activation of a particular type of glutamate receptor, the NMDA receptor (NMDAR), located on postsynaptic neurons in the dorsal horn of the spinal cord. Recently, a presynaptic NMDAR on the central terminals of extrinsic primary afferent neurons (EPANs) has been shown to regulate the release of substance P into the superficial dorsal horn, thereby contributing to central sensitization. It is likely that NMDAR subunits synthesized in DRG neurons of EPANs innervating the colon are transported not only to central, but also to peripheral nerve terminals. During the initial funding period of this grant, we have identified the NMDAR subunits expressed by EPANs and evaluated their functional properties. We found that activation of NMDARs in the colon acts to sensitize certain afferent terminals to mechanical distension. Using DRG neurons in primary culture, we showed that NMDAR activation enhances voltage gated calcium currents through a process that involves protein kinase C. Whether this mechanism accounts for the enhanced sensitivity of peripheral afferent nerve terminals to mechanical stimuli, and the nature of the intracellular transduction events coupling NMDAR to protein kinase C (PKC) and other downstream targets will be addressed in the renewal application. In addition, we showed that colon inflammation results in enhanced NMDAR signaling in DRG neurons, which is associated with phosphorylation of one of the NMDAR subunits. In this competitive renewal application, using a combination of in vivo (validated, targeted KO mice) and in vitro techniques (molecular biology, electrophysiology), we propose new experiments to address the following 3 hypotheses: 1) NMDARs on EPANs innervating the colon play a role in peripheral sensitization by regulating neuronal excitability and neuropeptide release. 2) Sensitization of EPAN terminals is mediated by the NMDAR through PKCepsilon and PKD activation. 3) Colon inflammation sensitizes DRG neurons via phosphorylation of NMDARs by non-receptor tyrosine kinases. These studies are likely to contribute significantly to our understanding of the mechanisms underlying enhanced visceral pain associated with functional and inflammatory disorders of the Gl tract.